It is commonly recognized that autoimmune diseases are provoked by activation of T cells reactive with self-antigens and cytokines or inflammatory mediators produced mainly by the activated T cells in turn damage the tissues. Thus, for treatment of autoimmune diseases, there have been attempts to suppress the activation of T cells reactive with self-antigens or to inhibit broadly and non-specifically the cytokines or the inflammatory mediators produced.
Recently, specific immunotherapies are expected to be the best therapy for autoimmune diseases. Once it becomes possible to identify peptide ligands recognized by every T cells at the peripheral, an antigenic epitope recognized by the T cells that are causing autoimmune diseases could be found out to lead to development of specific immunotherapies aimed at said etiologic T cells. For treatment of malignant tumors, it is also expected to develop specific immunotherapies wherein T cells that specifically attack tumor cells are chosen and activated. However, identification of antigens that are specific to patients and are recognized by T cells as well as determination of an amino acid sequence of said antigens has not yet been successfully achieved.
Much effort has been made for developing a method for identification of peptide ligands recognized by T cells. In case of identification of epitopes recognized by the previously reported T cell clones, a strategy has been employed wherein particular natural antigens to be anticipated were added during cloning of T cell and T cell clones responsive to said particular antigens were proliferated. This approach, however, is disadvantageous in that not T cell clones but those that recognize such particular antigens anticipated could be proliferated and if no T cells capable of recognizing said particular antigens are contained in the culture, then no proliferation of T cells could be obtained.
An alternative process for identifying peptide ligands recognized by cloned T cells have been reported (Tanaka, Y. et al., J. Immunol. 1999, 162: 7155–7161, “Identification of peptide superagonists for a self-K-ras-reactive CD4+ T cell clone by use of combinatorial peptide libraries and mass spectrometry”; Hemmer, B. et al., J. Exp. Med. 1997, 185: 1651–1659, “Identification of high potency microbial and self ligands for a human autoreactive class II-restricted T cell clone”).
However, as far as peripheral T cells of unknown specificity are concerned, clones per se could hardly be obtained to render it difficult to propagate T cells to a degree sufficient for testing. In order to obviate the difficulty, an attempt has been made to stimulate T cells with an immobilized anti-CD3 antibody in the presence of IL-2 but the long-term maintenance of T cells could not readily achieve. This is supposedly due to lack of physiological response of APC via APC-peptide-T cell interaction.
Thus, there is a need for developing a process for proliferating peripheral T cells of unknown specificity for cloning as well as for effective analysis of epitopes recognized by said T cells.
In the previous study, it was observed that some peptide partial agonists support T cell survival (Matsushita, S. et al., J. Immunol. 1997, 158: 5685–5691). It was also observed that an agonistic anti-CD29 antibody MAR4 increased the efficiency in establishing T cell clones from PBMC, both by suppressing antigen-driven activation-induced cell death and by enhancing the T cell proliferation, only in the presence of TCR/CD3-mediated stimulation (Tanaka, Y. et al., Hum. Immunol. 1998, 59: 343–351). Moreover, it was observed that culture supernatant of antigen-stimulated T cells in the presence of monocytes, increased the efficiency of cloning, when added to culture wells for limiting dilution.
Under the circumstances, the present inventors constructed combinatorial randomized peptide library and stimulated peripheral blood- or tissue-derived T cells with this library in the presence of an interleukin so that the T cells are proliferated and cloned. The isolated T cell clone is then analyzed for its epitope recognition to identify epitopes recognized by said clone by combinatorial assay with peptide library. Based on the identified peptide sequence, natural peptide ligands recognized by the isolated T cell clone can then be identified by pattern match search with data base of sequence.